With over 42 million people estimated to be infected with HIV-1 worldwide, there is a global urgency to develop a vaccine against the virus. It is becoming clear that neutralizing antibodies (Nabs) can provide effective prophylaxis against HIV-1 infection. However, eliciting Nabs that are broadly reactive against many different HIV-1 isolates has been a major challenge. This difficulty is largely due to the ability of the viral envelope glycoprotein to evade humoral immune response. In addition to extensive glycosylation and complex tertiary and quaternary structures, the envelope has 5 hypervariable regions that exhibit high degree of antigenic variation. Although variable loops V1/V2 and V3 are quite immunogenic and elicit Nabs, these antibodies are highly virus isolate-specific. A successful HIV-1 vaccine must be able to elicit Nabs that are broadly reactive. Therefore, it is important to continue to explore and evaluate new ideas, strategies and reagents. In this study, a novel combinatorial technology for producing long polynucleotides will be employed to generate a large library of envelope constructs with divergent variable loops. The long-term goal of the study is to evaluate biochemical and immunological properties of these envelope proteins. The specific aims of the study are: (1) to further characterize and to refine the process of generating random polynucleotide, (2) to generate envelope glycoproteins with antigenically diverse V3 loops and fusion-competent envelopes containing smaller V1/V2 loops, and (3) to develop a new DNA/viral vector vaccine strategy using T7 RNA polymerase-based expression system. Successful completion of this study will provide new reagents and strategies for eliciting potent humoral immune response, and would represent a major step forward in AIDS vaccine development efforts.